SLIC sub-cloning using T4 DNA polymerase treated inserts with RecA

doi:10.1038/nprot.2007.121

Method Article

SLIC sub-cloning using T4 DNA polymerase treated inserts with RecA

https://doi.org/10.1038/nprot.2007.121

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Genetics

homologous recombination

SLIC

iPCR

RecA

We describe a novel cloning method SLIC that allows the assembly of multiple DNA fragments in a single reaction using in vitro homologous recombination and single-strand annealing.

Digest 2 μg of vector with restriction enzymes. Gel purify the vector and isolate the DNA using QIAEX II gel extraction kit. Quantitate the vector.
Amplify inserts using Taq DNA polymerase. Set up a 100 μl PCR reaction with 250 μM of each dNTP, 0.5 μM of each primer, and 2.5 U of Taq DNA polymerase (from Eppendorf). Cycle as follows: 94°C for 45 seconds; 30 cycles of 94°C for 45 seconds, 54°C for 45 seconds, and 72°C for 1 minute; 72°C for 10 minutes. Add 20 U of DpnI to 100 μl of PCR products after PCR, incubate at 37°C for 1 hour (not necessary if going from a MAGIC vector to ColE1 origin). Purify the PCR products by QIAquick PCR purification column. Quantitate the inserts. We typically have 20 bp homology between the vector and the inserts.
Take 1 μg of the vector and 1 μg of the inserts and treat separately with 0.5 U of T4 DNA polymerase in T4 buffer (NEB) plus BSA in a 20 μl reaction at room temperature for 30 minutes. Stop the reaction by adding 1/10 volume of 10 mM dCTP and leave on ice.
Set up a 10 μl annealing reaction using 1:1 insert to vector ratio with 3 ng or less of a 3.1 kb vector (0.0015 pmol), 1x ligation buffer (NEB), appropriate amount of insert, 20 ng of RecA protein (Epicentre Biotechnologies) and water. Incubate at 37°C for 30 minutes. Leave on ice or store in -20°C.
Add 5 μl of the annealed mixture into 150 μl of BW23474 chemical competent cells, incubate on ice for 30 minutes, heat shock at 42°C for 45 seconds, return to ice for 2 minutes, add 0.9 ml of SOC and recover at 37°C for 1 hour.
Plate 100 μl onto plates containing the appropriate antibiotics and incubate at 37°C overnight. (Cl-Phe is used for vectors that have PheS-G294 between restriction enzyme sites. We have found that most background comes from uncut vector in our preps and therefore we can select against it with Cl-Phe. However, Cl-Phe is not an essential step and usually only has a 2-fold effect on background.)

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SLIC sub-cloning using T4 DNA polymerase treated inserts with RecA

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